We propose to provide the Drosophila research community with new genetic tools that will allow an unprecedented level of designer mutation and manipulation of the genome in vivo. First, we will create a collection of 6,500 D. melanogaster strains carrying single insertions of a novel transposable element named Mi{MIC}, distributed throughout the genome. Mi{MIC} contains two phage fC31 attP sites for site-specific recombination. After insertion of Mi{MIC} in the genome, any segment of DNA up to 120 kb can be readily integrated into the genome at the site of the Mi{MIC} element. Second, we will create a collection of tagged fusion protein alleles for selected genes. One-third of Mi{MIC} insertions are in coding introns of genes, and Recombination Mediated Cassette Exchange can be used to efficiently swap in DNA cassettes between the paired attP sites in such Mi{MIC} insertions to create tagged gene alleles. We have created a battery of cassettes that can be used to 1) monitor spatial and temporal patterns of protein expression by antibody staining or fluorescent imaging of live animals, 2) map transcription factor binding sites using chromatin immunoprecipitation, 3) identify protein interactions by immunoprecipitation-mass spectrometry, or 4) create temperature-sensitive mutations. We will generate protein fusions in 1,000 genes of interest using this strategy. Mi{MIC} insertions can also be used to generate precisely targeted mutations at a distance of up to 80 kb from the insertion site. We estimate that 95% of Drosophila genes will be within 20 kb of one of the Mi{MIC} insertions and therefore well within range for targeted mutagenesis. A nearby Mi{MIC} can be used to make a precise deletion of the gene, introduce point mutations for structure-function analysis, or introduce regulatory mutations that allow inducible spatial or temporal mis-expression of the gene. In addition, researchers can use Mi{MIC} insertions to make insertions, deletions, or point mutations of non-coding genetic elements such as RNA genes, chromatin boundary elements and origins of replication. The Mi{MIC} collection will be made publicly available and will enable researchers to manipulate almost all fly genes at an unprecedented speed and level of sophistication. PUBLIC HEALTH RELEVANCE: Basic research in the model organism, Drosophila melanogaster, has significantly helped us in providing a much better understanding of the biology of higher organisms such as humans, largely due to the evolutionary conservation of genetic pathways. Many genes discovered in Drosophila have been linked to human diseases including cancer, neurodegenerative diseases, diabetes, and inherited developmental diseases. The elucidation of biological processes in the fly have greatly influence our understanding of these diseases. Hence, the creation of mutant strains and other genetic tools proposed here will be a highly valued resource to the Drosophila research community and will greatly increase the efficiency of basic and translational research in many labs.